During the tenure of this application our objectives are as follows: 1) To generate phospho state-and site- specific antibodies against synthetic phospho and dephospho peptides containing a sequence of amino acid (AA) surrounding serine (Ser)40, Ser31, Ser19 in tyrosine hydroxylase (TH) and to determine their ability to distinguish between phospho and dephospho states and sites of the enzyme. We have generated polyclonal antibodies which recognize the phospho site of TH at Ser40, anti-pTH32-47 (initially designated as anti-pTH16), and we will further characterize the ability of these antibodies to distinguish between phospho and dephospho states and sites of TH in NE and DA containing brain regions. The findings that inhibition of PP activity is required for antipTH32-47 to recognize striatal TH on Western blots and immunohistochemically provides the basis for our studies on phospho/dephospho states of TH in DA and NE containing brain regions. Our ultimate goal is to utilize these antibodies for measuring the phospho/dephospho states of TH in response to various physiological and pharmacological manipulations; 2) To investigate the activation and phosphorylation of TH elicited by treatment with antipsychotic drugs (APD). The mechanisms by which acute administration of APD elicits activation and protein kinase-mediated phosphorylation of TH, while chronic elicits deactivation and dephosphorylation of the enzyme will be investigated. Rhospho/dephospho state- and site-specific antibodies against segments of TH will be used to determine the phosphorylation states of the enzyme at a Ser site in specific brain regions following acute and chronic treatment with APD. Immunohistochemical studies will elucidate the ratio of phospho/dephospho TH in specific neuronal populations prior to and following acute and chronic treatments with APD; 3) To study the mechanisms underlying the activation of TH by site-directed mutagenesis at Ser40 and to elucidate the interactions between Ser40, Ser31 and Serl9 of TH. We will elucidate the mechanisms underlying the enhanced TH activity of THSer40 mutant (THSer40m) in which Ser40 was substituted with Leu or Tyr. It will be assessed to what extent the enhanced formation of L-dopa catalyzed by THSer40m is due to the diminished inhibition of the enzyme by end products and to changes in the phosphorylation patterns of TH at multiple Ser sites. The effects of mutation at Ser40, Ser31, and Ser19 on phosphorylation of TH at multiple Ser sites of the enzyme catalyzed by protein kinase A (RKA), PKC and by Ca++/calmodulin protein kinase II (Ca++/CaMpKII) will be determined.